Sodium butyrate and hydroxyurea, effective inhibitors of DNA synthesis in HeLa cells, cause these cells to produce increased levels of the ectopic glycopeptide hormones human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), and free a chains for these hormones. The objective of this study was an assessment of the role of modulation of cell cycle events in the action of these two chemical agents. A variety of experimental approaches was employed to obtain a clear view of the drugs' effects on cells located initially in all phases of the cell cycle. Cells in early GI, G,, or M phase a t time of addition of either inhibitor were not arrested a t early time points, but by 48 hours became collected a t a location characteristic for each drug, near the GI-S phase boundary. Flow microfluorometry (FMF) and thymidine labeling index revealed that butyrate-treated cells arrested late in G I phase very close to S phase, while hydroxyurea-blocked cells continued to early S phase. Both inhibitors prevented cells originally in S phase from reaching mitosis. S cells exposed to hydroxyurea were killed by 48 hours, but those growing in 5 mM butyrate progressed to the end of S or G, phase where they became irrever-